I. Field of the Invention
The present invention relates to the fields of molecular biology and medicine. More specifically, the invention relates to methods and compositions for the treatment of diseases or conditions that are affected by microRNA (miRNA) miR-124 expression or lack thereof, and genes and cellular pathways directly and indirectly modulated by such.
II. Background
In 2001, several groups used a cloning method to isolate and identify a large group of “microRNAs” (miRNAs) from C. elegans, Drosophila, and humans (Lau et al., 2001; Lee and Ambros, 2001; Lagos-Quintana et al., 2003). Several hundreds of miRNAs have been identified in plants and animals—including humans—which do not appear to have endogenous siRNAs. Thus, while similar to siRNAs, miRNAs are distinct.
miRNAs thus far observed have been approximately 21-22 nucleotides in length, and they arise from longer precursors, which are transcribed from non-protein-encoding genes (Carrington and Ambros, 2003). The precursors form structures that fold back on themselves in self-complementary regions; they are then processed by the nuclease Dicer (in animals) or DCL1 (in plants) to generate the short double-stranded miRNA. One of the miRNA strands is incorporated into a complex of proteins and miRNA called the RNA-induced silencing complex (RISC). The miRNA guides the RISC complex to a target mRNA, which is then cleaved or translationally silenced, depending on the degree of sequence complementarity of the miRNA to its target mRNA. Currently, it is believed that perfect or nearly perfect complementarity leads to mRNA degradation, as is most commonly observed in plants. In contrast, imperfect base pairing, as is primarily found in animals, leads to translational silencing. However, recent data suggest additional complexity (Bagga et al., 2005; Lim et al., 2005), and mechanisms of gene silencing by miRNAs remain under intense study (Chendrimada et al., 2007; Kiriakidou et al., 2007).
Recent studies have shown that changes in the expression levels of numerous miRNAs are associated with various cancers (reviewed in (Calin and Croce, 2006; Esquela-Kerscher and Slack, 2006; Wiemer, 2007). miRNAs have also been implicated in regulating cell growth and cell and tissue differentiation—cellular processes that are associated with the development of cancer.
The inventors previously demonstrated that hsa-miR-124 is involved with the regulation of numerous cell activities that represent intervention points for cancer therapy and for therapy of other diseases and disorders (U.S. patent application Ser. No. 11/141,707 filed May 31, 2005 and Ser. No. 11/273,640 filed Nov. 14, 2005, each of which is incorporated herein by reference in its entirety). For example, cell proliferation, cell division, and cell survival are frequently altered in human cancers. Transfection of human lung carcinoma cells (A549) and human cervical cancer cells (HeLa) with synthetic hsa-miR-124 reduced viable cell numbers. In addition, the inventors showed that miR-124 significantly increased the capacity of two therapeutic compounds (TRAIL, an apoptosis pathway activator in cancer cells, and etoposide, a topoisomerase II inhibitor that activates the apoptosis pathway in cancer cells and normal cells) to induce cell death in A549 or HeLa cells. Overexpression of synthetic miR-124 in various cell lines decreased cell proliferation. In those studies, the inventors observed reduced proliferation of human breast cancer cells, (BT549), normal human breast epithelial cells (MCF12A), human cervical cancer cells (HeLa), human prostate carcinoma cells (22RV1), human basal cell carcinoma cells (TE 354.T), normal human skin cells (TE 353.Sk), and human lung carcinoma cells (A549, CRL-5826, HTB-57). Overexpression of miR-124 in HeLa cells significantly reduced the number of cells in the G2/M phase of the cell cycle when compared to cells transfected with a negative control miRNA. Also, others have recently observed that epigenetic silencing of miR-124a in cancers cells modulates activity the oncogene, CDK6 and the tumor suppressor gene, Rb (Lujambio et al., 2007).
Bioinformatics analyses suggest that any given miRNA may bind to and alter the expression of up to several hundred different genes. In addition, a single gene may be regulated by several miRNAs. Thus, each miRNA may regulate a complex interaction among genes, gene pathways, and gene networks. Mis-regulation or alteration of these regulatory pathways and networks involving miRNAs are likely to contribute to the development of disorders and diseases such as cancer. Although bioinformatics tools are helpful in predicting miRNA binding targets, all have limitations. Because of the imperfect complementarity with their target binding sites, it is difficult to accurately predict the mRNA targets of miRNAs with bioinformatics tools alone. Furthermore, the complicated interactive regulatory networks among miRNAs and target genes make it difficult to accurately predict which genes will actually be mis-regulated in response to a given miRNA.
Correcting gene expression errors by manipulating miRNA expression or by repairing miRNA mis-regulation represent promising methods to repair genetic disorders and cure diseases like cancer. A current, disabling limitation of this approach is that, as mentioned above, the details of the regulatory pathways and gene networks that are affected by any given miRNA, have been largely unknown. This represents a significant limitation for treatment of cancers in which a specific miRNA may play a role. A need exists to identify the genes, genetic pathways, and genetic networks that are regulated by or that may regulate expression of miRNAs.